This invention is directed to chromatographic media and method for direct processing of crude feeds on columns for isolation of biologically active materials.
Traditionally, downstream processing of biologics from cell culture/fermentation harvests has required two major operations: recovery and purification. Recovery involves the removal of cellular and other particulate materials by centrifugation and/or microfiltration, as well as an initial volume reduction step, typically ultrafiltration. Since conventional chromatography media are rapidly fouled by cell debris, particle-free feed must be prepared for the purification operation.
Centrifugation and filtration are not only lengthy and costly operations, they compromise quality. Proteases released from broken cells can degrade the target protein, further complicating the task of purification method development and increasing purification costs. The longer the contact time with the concentrated cellular debris, the more product may be lost.
Direct capture of the protein product from the unclarified feed would minimize product degradation and improve product quality, yield and process economy. Also, the capital-intensive recovery operation would be greatly simplified if the product capture and cell removal steps were combined into a single operation.
There are two approaches to directly capture product from unclarified feed, such as cell culture/fermentation harvest or other biological sample (e.g., blood plasma). One proposes fluidization of the capture resin particles. Via fluidization, the individual particles are separated so that the debris can exit the column bed unobstructed.
This approach suffers from several problems. The product breaks through the fluidized bed early on due to channelling, causing product loss. The fluidized bed system operates at a predetermined low flow rate, i.e., there is no flexibility in the operation or means for changing the size of the column. The buffer consumption of the system is higher than on packed bed systems, which is a significant cost factor for high value pharmaceutical products, many of which require specialized buffers for their purification. Fluidized bed operation also requires specialized, costly hardware and chromatography media.
The other approach is the use of packed bed columns for particulate removal. This avenue has remained unexplored for the following reason. To clear cellular debris on a packed bed column requires using large, preferably spherical particles. These particles require sufficient space in the interparticle lumen to let cells or other particulates of comparable size to exit the column.
The downside of using large particles (beads) is that the protein binding capacity is a function of the available surface per unit volume of gel bed. Therefore with increased particle diameters a loss of binding capacity is observed. When the particle diameter is increased from 0.1 mm to 1 mm, such as is required to handle dense cell suspensions, approximately 90% of the protein binding capacity is lost. This made packed bed columns impractical for processing crude process feed streams.
Packed bed column operation, however, offers simplicity, efficiency and economy. It is flexible and easy to scale. There is no need for specialized particles, equipment or training of the operators. The production floor-space is relatively small for standard chromatography and there is no need for the modification of the height of the production facility to accommodate the fluidized bed equipment.
Product application rate is another important issue in terms of throughput of the operation. This is predetermined for fluidized bed systems, but for packed column systems just the reaction binding kinetics is the rate limiting factor. This allows higher throughput, up to 3-10 times higher than for fluidized bed systems.
Crude biological feed streams often have a physiological ionic strength which is too high in many situations to allow direct binding of the protein of interest to ion exchange media. Therefore, dilution of the feed with deionized water is required, typically 3-5 times. This greatly increases the total volume of the feed to be processed and partially offsets the advantage of direct processing versus the traditional centrifugation-filtration-concentration route. Ionic strength reduction in the crude feed stream other than dilution is therefore desirable.
An alternative method for reducing ionic strength is dialysis. However, dialysis is impractical with unclarified feed streams as particulate matter will rapidly clog dialysis membranes. Therefore, with current technologies the only practical method for ionic strength reduction in crude feed streams is dilution.
There is therefore a need for improved chromatographic materials and methods to achieve direct processing of crude feeds, such as cell culture/fermentation harvests tissue extracts, or blood plasma, on packed bed columns. There is also a need for ion removal from crude samples without dilution and the resulting loss of throughput.